Proper regulation and expression of major histocompatibility complex class II (MHC-II) genes is critical to developing and maintaining an active and healthy immune system. The goals of this program are to understand the molecular mechanisms responsible for their regulation. In particular this research program seeks to understand the role of chromatin in the regulation of MHC-II genes. We present data that show that the current paradigm whereby the proximal WXY regulatory motifs, which bind RFX, CREB, NF-Y and CIITA is incomplete in two ways. The first is that the paradigm does little to understand the relationship between chromatin modifications at MHC-II promoters and expression. The master regulator of MHC-II transcription CIITA coordinates most of the chromatin modifications at MHC-II promoters. However, little is known about how this occurs. Aim 1 seeks to determine: the timing of the modifications, the relationships between the remodeling factors and CIITA, and what role the modifications have on MHC-II gene expression. Secondly, the paradigm does not include elements that regulate or coordinate expression through changes in nuclear architecture or structure. We provide evidence that we have discovered one such element. A region termed XL9, located in the intergenic space between HLA-DRB1 and HLA-DQA1, does not possess enhancer function, but is highly acetylated, interacts with the nuclear matrix, functions as an enhancer block, and is the nexus of two long range chromatin loops with the promoter regions of the above HLA genes. Moreover, XL9 binds CCCTC transcription factor (CTCF) a factor associated with chromatin insulator function and genomic imprinting. Knockdown of CTCF activity reduces expression of HLA-DRB1. The data suggest that XL9 functions to separate and coordinate the expression of the flanking MHC-II genes. The HLA-DRB1 and HLA-DQA1 genes are the first immune system genes known to be regulated by CTCF. Aim 2 will characterize the exact role of XL9 in regulating MHC-II expression. CIITA was required for interactions with XL9 and preliminary data suggests that CIITA interacts with CTCF. We propose to examine these interactions and identify the factors associated with this complex. Such components are likely to link the biological activities of chromatin remodeling events observed with the biology of MHC-II regulation. While extremely novel in its activity, we suggest that other XL9-like elements will regulate MHC-II genes. Aim 3 is designed to identify additional MHC-II promoter interactions and XL9-like elements with in the MHC-II region. The results from this work should provide a comprehensive view of the chromatin dynamics of MHC-II regulation and provide novel mechanisms of gene control that will be applicable to many immune system genes. Such mechanisms should provide novel targets for future immune based therapies.